a) Field of the Invention
The invention is directed to an optical method for the targeted transfer of molecules, preferably DNA, RNA, peptides, amino acids and proteins, into vital cells by means of laser radiation and to an arrangement for implementing the method.
The method is advantageously suitable for the transfection of plant cells, animal cells and human cells, for example, for producing drugs such as synthetic vaccines.
By means of the arrangement according to the invention, the method can be used effectively for the transfection of genes in individual cells and opens up applications in the fields of plant materials and animal materials production, gene therapy and in the production and application of specific drugs, particularly synthetic vaccines.
b) Description of the Related Art
Targeted molecule transfer plays an important role in the production of vaccines, among other things. Vaccines are used within the framework of active immunization of humans and animals for stimulating the immune system against pathogenic microorganisms and pathogenic substances. Normally, inactivated or attenuated germs which still retain an immunogenic effect are used. A slight health risk is involved in activating individual attenuated germs. This may have fatal consequences particularly for patients with weakened immune defense. Considerably more critical, however, is the fact that there is currently no vaccine available for many diseases, including AIDS. Methods in gene technology aim at making possible the production of effective, highly-pure synthetic vaccines through DNA transfer. The implantation of foreign DNA in plants and animals for expressing vaccines in foodstuffs is being discussed. In principle, a specific immunity against very particular amino acid sequences can be achieved.
The transporting of foreign DNA to a target cell can be carried out by means of specific carriers, e.g., colloidal particles such as nanospheres and microspheres, emulsions and liposomes. Virus-like aggregates (VLA) in which the molecules to be transported are enclosed by a two-layer membrane are currently being researched.
When colloidal particles of this kind are applied intravenously, for example, they are normally intercepted with high efficiency by the reticuloendothelial system (Kupffer cells, etc.) and, therefore, cannot effect the transfer to the actual target. In order to prevent this unspecific binding, the surface of the particles is changed, e.g., through determined coatings and suitable particle sizes, by means of complicated particle engineering. Finally, when the target cell in the target organ is reached more or less effectively and selectively, problems arise with respect to particle reception and there is a risk of destruction through lysosomal enzymes and nucleases.
Therefore, an efficient foreign DNA accumulation in the cytoplasm or directly in the cell nucleus of a specific target cell is desirable.
The targeted transfer of molecules, preferably of DNA into vital cells, was carried out heretofore:
(i) by mechanical processes such as microinjection and particle gun bombardment;
(ii) by means of biological (viruses, bacteria, etc.) and synthesized carrier molecules; or
(iii) by permeabilizing the membrane by means of electrical fields (electroporation) or chemical agents (e.g., streptolysin O toxin).
There are problems with all three of these methods with respect to the efficiency of the molecule transfer and the high probability of an unintended lethal effect as is confirmed by the prior art mentioned in the following.
It is known (e.g., D. J. Stephens, R. Pepperkok: The many ways to cross the plasma membrane, Proc. Nat. Acad. Sci. USA, 89 (2001) 4295-4298; D. Luo, W. M. Saltzman: Synthetic DNA delivery systems, Nature Biotechnol. 18 (2000) 33-37; L. Bildirici, P. Smith, C. Tzavelas, E. Horefti, D. Rickwood: Transfection of cells by immunoporation, Nature 405 (2000) 298) to carry out a transfer of molecules, preferably of DNA, into vital cells by means of synthesized carrier molecules and biological carrier systems (viruses, etc.).
It is likewise known to enable the targeted transfer of molecules through mechanical methods such as microinjection and particle gun bombardment (e.g., M. Knoblauch et al.: A galinstan expansion femtosyringe for microinjection of eukaryotic organelles and prokaryotes, Nature Biotechnol. 17 (1999) 906-909).
Further, the transfer of molecules into vital cells by permeabilization of the membrane by means of electrical fields (electroporation) or chemical agents (e.g., streptolysin O toxin) is known.
In conventional vaccine production using gene technology, cell cultures are typically infected with viruses, as carriers of the DNA in question, under strict safety measures in bioreactors; the viruses are then inactivated or attenuated.
The direct transfer of individual molecules in a specifically selected individual cell is only possible by means of mechanical microinjection using thin glass cannulas (typical distal diameter: 0.5 mm) through invasive disruption of the cell membrane; this process is inefficient and entails a high potential of injury. Mechanical gene transfer by manual microinjection requires specially trained personnel and is met by considerable difficulties when transferring into nonadherent cells, in plant cells because of the sturdy cell wall, and with isolated protoplasts. In addition, the presence of the glass in the interior of the cell causes considerable problems due to the adherence of intracellular molecules, e.g., certain proteins. The occurring mechanical forces result in additional destructive effects.
Optical methods for targeted molecule transfer based on focused laser radiation through the microablation of a membrane section were carried out heretofore by means of ultraviolet laser sources with pulse widths in the nanosecond range and high energy in the microjoule and millijoule range. Laser pulses with such long pulse widths generate collateral destructive mechanical effects through intensive photodisruptive processes. In addition, the application of ultraviolet radiation is controversial due to cytotoxic and mutagenic effects. UV radiation is also absorbed outside the focus area by a plurality of endogenic molecules. Accordingly, the success rate of this type of optical transfection is low. Testing of laser-assisted gene transfer has been conducted since 1984 (Tsukakoshi et al. (1984) Appl. Phys. B35 135-140). Normally, ultraviolet (UV) nanosecond lasers with a relatively high pulse energy in the μJ range and single-shot mode are used. The transfection rates are very low, as is confirmed in the literature by Tsukakoshi et al. (Appl. Phys. B35 (1984) 135-140) and Kurato et al. (Exp. Cell Res. 162 (1986) 372-378) which shows transfection efficiencies of a maximum 0.6%. Tsukakoshi et al. describe an arrangement for the transfer of genes which is based on a frequency-tripled 10 Hz Nd:YAG nanosecond laser at a working wavelength of 355 nm and which is outfitted, in addition, with a He—Ne laser as pilot laser and enables beam deflection through the use of two galvoscanners. An image of the sample is displayed on a TV monitor by means of a transillumination apparatus using an UV blocking filter and is recorded by means of video recorders. Single shots with high pulse energies of 1 mJ were used for perforating the cell membrane.
Nitrogen lasers with an emission wavelength of 337 nm were also used for gene transfer in plant cells (Weber et al.: Naturwissenschaften 75 (1988) 36). In UV laser-assisted gene transfer in plant embryos, a transformation efficiency of 0.5% was reported (Greulich: “Micromanipulation by light in biology and medicine”, Birkhä user Verlag, 1999).
Another publication by Tao et al. (PNAS 84 (1987) 4180-4184) describes UV exposure with μJ pulses with a pulse duration of 10 ns in a relatively large irradiation area of 2.0-μm diameter using an inverted Zeiss microscope with a 32× objective. At this magnification, the numerical aperture is typically less than 0.8. The relatively large irradiation spot involved probably caused a considerable perforation in the membrane on the same order of magnitude. The transformation efficiency in these tests using human cells was less than 0.3%.
In sum, it can be stated that there are no known methods or apparatus usable in practice for transferring molecules, particularly for the production of synthetic vaccines, into a specially selected individual cell efficiently and effectively without damaging the living cell.